The causative agent of the fatal disease Tularemia, Francisella tularensis (Ft), is a gram negative highly infectious intracellular bacterium, that is classified as a Category A Select Bioterrorism Agent. Intracellular replication of Ft within macrophages is essential for disease manifestation. It is rather astonishing the relative paucity of knowledge about the molecular and cellular aspects of pathogenesis of this versatile and extremely virulent pathogen. Our preliminary data indicate that upon entry into macrophages, Ft-containing phagosome (FCP) evades lysosomal fusion. The organism escapes from the phagosome into the cytoplasm by 2-12h post-infection. Our hypothesis is that specific modes of entry of Ft into macrophages allow the organism to modulate biogenesis of its phagosome, by specific exported bacterial effectors, into a niche that does not fuse to the lysosomes and allow subsequent escape of the organism into the cytoplasm, where it replicates. To test this hypothesis, our specific aims are: I. To dissect the cell biology of intracellular F. tularensis and the unique mechanisms that halt phagosome biogenesis at a non-acidified late endosome-like stage. II. To identify mutants defective in arresting phagosome biogenesis and escape into the cytoplasm. III. To characterize the bacterial factors involved in arresting phagosome biogensis and escape into the cytoplasm. Significance: Pathogenesis of the Category A Select Bioterrorism Agent Ft is one of the least studied and understood among intracellular bacterial pathogens. Understanding how this organism exploits macrophages for proliferation and disease manifestation is fundamental to our knowledge of tularemia. The bacterial effectors involved in exploiting the macrophage are potential targets for treatment as well as potential vaccine candidates. [unreadable] [unreadable] [unreadable]